Mechanism of Meiotic Recombination

减数分裂重组机制

• 批准号: 7592518
• 负责人: MICHAEL J LICHTEN
• 金额: $ 123.31万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7592518
• 关键词: Area; Biological Process; Bloom Syndrome; Chromosome Segregation; Chromosome Structures; Chromosomes; Class; Complex; DNA Double Strand Break; DNA Repair; Data; Double Strand Break Repair; Elements; Event; Frequencies; Genes; Genetic Crossing Over; Genetic Recombination; Genome; Growth; Homologous Gene; Investigation; Location; Meiosis; Meiotic Recombination; Metabolism; Methods; Mitotic Cell Cycle; Molecular; Mutate; Pathway interactions; Play; Ploidies; Proteins; Research; Resolution; Role; Saccharomyces cerevisiae; Saccharomycetales; Structure; System; Techniques; Time; Up-Regulation; Yeasts; base; endonuclease; helicase; human PLK1 protein; member; mutant; novel; prevent; repaired; research study

Progress has been made in the following areas: Initiation of meiotic recombination: Meiotic recombination is initiated by DNA double-strand breaks (DSBs); both the location and timing of break formation is tightly controlled. Our aim is to determine the substrate requirements of proteins that form DSBs, and the factors that control their location, frequency and timing. Current research is directed at determining the chromosome structural elements that determine where DSBs do and do not form. We developed a novel technique to isolate intermediates in double-strand break-repair, based on their partially single-stranded DNA content. We applied this method to a microarray-based whole genome analysis to recombination intermediate distributions, and have determined the true distribution of meiotic recombination initiation events across the genome. Our data show that meiotic DSBs are much more evenly distributed than had been previously believed, and also illuminate the mechanism of DSB formation and repair during meiosis. Mechanism of meiotic recombination: We have developed techniques to isolate and characterize unstable intermediates in meiotic recombination, and have used these techniques to demonstrate that the two classes of meiotic recombination (events associated with crossing-over <I>versus</i> events not accompanied by crossing-over) proceed by distinct molecular mechanisms. Our current aim is to determine the repair proteins that participate in both pathways, that are unique to one or the other pathway, and that determine the choice between crossover and noncrossover recombination. Recent experiments identify the Sgs1 helicase (the budding yeast homolog of the helicase mutated in Bloom's syndrome) and the Mus81/Mms4 structure-specific endonuclease as playing a critical role in controlling meiotic recombination. In the absence of these two proteins, abnormal recombination intermediates accumulate and block chromosome segregation. Using a system that allows induced expression of target proteins late in meiosis, we showed that Mus81/Mms4 is resolves these abnormal intermediates after they are formed, while Sgs1 prevents their formation. Because mutants lacking both Sgs1 and Mus81/Mms4 are inviable, it is likely that these two proteins play similar roles in chromosome metabolism during the mitotic cell cycle. This subject is a subject of current investigation. We previously had shown that upregulation of a class of meiotically-expressed genes (middle-meiosis genes) was a critical step in the resolution of recombination intermediates as crossovers. We have now identified Cdc5, the budding yeast polo-like kinase, as the sole member of the >200 middle-meiosis genes that is required for both crossover formation and for the disassembly of meiosis-specific chromosome structures in advance of the meiotic divisions. Identification of the functional Cdc5 target is underway.

减数分裂重组的启动：减数分裂重组是由DNA双链断裂（DSB）启动的；断裂形成的位置和时间都受到严格控制。我们的目标是确定形成 DSB 的蛋白质的底物要求，以及控制其位置、频率和时间的因素。目前的研究旨在确定决定 DSB 形成和不形成位置的染色体结构元件。我们开发了一种新技术，根据部分单链 DNA 含量分离双链断裂修复中间体。我们将该方法应用于基于微阵列的全基因组分析以重组中间分布，并确定了减数分裂重组起始事件在整个基因组中的真实分布。我们的数据表明，减数分裂 DSB 的分布比之前认为的要均匀得多，并且也阐明了减数分裂过程中 DSB 形成和修复的机制。减数分裂重组机制：我们开发了分离和表征减数分裂重组中不稳定中间体的技术，并使用这些技术来证明两类减数分裂重组（与交叉<I>与</i>事件相关的事件）伴随着交叉）通过不同的分子机制进行。我们当前的目标是确定参与这两种途径的修复蛋白，这些修复蛋白对于一种或另一种途径是独特的，并决定交叉重组和非交叉重组之间的选择。最近的实验发现 Sgs1 解旋酶（布卢姆综合征中突变的解旋酶的出芽酵母同源物）和 Mus81/Mms4 结构特异性核酸内切酶在控制减数分裂重组中发挥着关键作用。在缺乏这两种蛋白质的情况下，异常重组中间体会积累并阻止染色体分离。使用允许在减数分裂后期诱导表达靶蛋白的系统，我们发现 Mus81/Mms4 可以在这些异常中间体形成后解决它们，而 Sgs1 可以阻止它们的形成。由于同时缺乏 Sgs1 和 Mus81/Mms4 的突变体是无法存活的，因此这两种蛋白质很可能在有丝分裂细胞周期中的染色体代谢中发挥相似的作用。该主题是当前调查的主题。我们之前已经证明，一类减数分裂表达基因（减数分裂中期基因）的上调是解决重组中间体交叉的关键步骤。我们现已鉴定出芽酵母 polo 样激酶 Cdc5 是超过 200 个减数分裂中期基因的唯一成员，该基因是交叉形成和减数分裂之前减数分裂特异性染色体结构分解所必需的。功能性 Cdc5 靶标的识别正在进行中。